Numerous recent advances in the study of biology have benefited from improved methods of analysis and sequencing of nucleic acids. For example, the Human Genome Project has determined the entire sequence of the human genome which is hoped to lead to further discoveries in fields ranging from treatment of disease to advances in basic science. While the “human genome” has been sequenced there are still vast amounts of genomic material to analyze, e.g., genetic variation between different individuals, tissues, additional species, etc.
Devices for DNA sequencing based on separation of fragments of differing length were first developed in the 1980s, and have been commercially available for a number of years. However, such technology involves running individual samples through capillary columns filled with polyacrylamide gels and is thus limited in throughput due to the time taken to run each sample. A number of new DNA sequencing technologies have recently been reported that are based on the massively parallel analysis of unamplified (WO00006770: Proceedings of the National Academy of Sciences U.S.A. 100, 3960-3964 (2003)) or amplified single molecules, either in the form of planar arrays (WO9844151) or on beads (WO04069849: Nature, 437, 376-380 (2005): Science, 309, 5741, 1728-1732 (2005); Nat Biotechnol. 6, 630-6344 (2000)).
The methodology used to analyze the sequence of the nucleic acids in such new sequencing techniques is often based on the detection of fluorescent nucleotides or oligonucleotides. The detection instrumentation used to read the fluorescence signals on such arrays is usually based on either epifluorescence or total internal reflection microscopy, for example as described in WO9641011, WO00006770 or WO02072892. Whilst total internal reflection microscopy has been used to image both single and amplified molecules of DNA on surfaces, a robust, reliable, four color DNA sequencing platform (e.g., comprising heating systems, fluidic controls, uniform illumination, control of the optical beam shape, an autofocus system, and hill software control of all components) is described herein for the first time.
There is a continuing need for better, more robust, and more economical devices and systems for fast reliable sequencing of nucleic acids. The current invention provides these and other benefits which will be apparent upon examination of the current specification, claims, and figures.